This invention relates to diagnosis of Lyme disease. More particularly, this invention relates to improved serodiagnostic performance for Lyme disease by use of two recombinant proteins in ELISA as compared to standardized two-tier testing.
Lyme disease (LD) continues to be the most common vector-borne disease in North America, Hyde J A, Borrelia burgdorferi Keeps Moving and Carries on: A Review of Borrelial Dissemination and Invasion, Front. Immunol. 8:114 (2017), with increasing incidence in the United States and Canada, Waddell L A, Greig J, Mascarenhas M, Shannon H, Lindsay R, Ogden N, The Accuracy of Diagnostic Tests for Lyme Disease in Humans, A Systematic Review and Meta-Analysis of North American Research. PLOS ONE. DOI:10.1371/journal.pone.0168613 (2016). The primary agent of LD in North America is the spirochete Borrelia burgdorferi sensu stricto which is transmitted chiefly by the tick vectors Ixodes scapularis and I. pacificus, Shapiro E D, Borrelia burgdorferi (Lyme Disease), Pediatr. Rev. 35:500-509 (2014). The diagnosis of LD is based on patient history, clinical presentation, and serology, Hyde, supra; Shapiro, supra. The observation of erythema migrans (EM), which is present in 70-80% of early LD patients, Id., is very important in the detection and diagnosis of this stage of disease and is considered pathognomonic in endemic areas, Seriburi V, Ndukwe N, Chang Z, Cox M E, Wormset G P, High Frequency of False Positive IgM immunoblots for Borrelia burgdorferi in Clinical Practice, CMI Clin. Microbiol. Infec. 18:1236-1240 (2012). Direct detection of the spirochete, either by culture or by PCR amplification of Borrelia genes, is often unreliable due to the small number of organisms present in any sample or stage of infection. Waddell, et al., supra; Shapiro, supra. Therefore, serology remains the most important confirmatory step in the diagnosis of LD. Theel E S, The Past, Present, and (Possible) Future of Serologic Testing for Lyme Disease, J. Clin. Microbiol. 54:191-1196 (2016). As it is with many infectious diseases, early detection and confirmation of LD is difficult to establish, but it is crucial in the management of the disease. Early treatment for LD can mitigate or even prevent the complications of late stage illness, which can adversely affect the heart, nervous system, and joints and can persist for months or even years. Strie K, Jones K L, Drouin E E, Li X, Steere A C, Borrelia burgdorferi RST1 (OspC Type A) Genotype is Associated with Greater Inflammation and More Severe Lyme Disease, Am. J. Pathol. 178:2726-2739 (2011).
The most reliable serological testing method for the diagnosis of LD, which was recommended by the Center for Disease Control and Prevention (CDC) in 1995, CDC, Recommendations for test performance and interpretation from the second national conference on serologic diagnosis of Lyme disease, Morbidity and Mortality Weekly Report 44:590-591 (1995), continues to be a 2-tiered test method. The first-tier is a screening assay, and is most commonly an enzyme-linked immunosorbent (ELISA) assay, using as the antigens either a whole cell lysate of B. burgdorferi or specific recombinant proteins or peptides. Johnson B J B, Laboratory Diagnostic Testing for Borrelia burgdorferi Infection, CAB International 73-87 (2011). It is typically of low specificity but high sensitivity. If the first-tier test is negative, the sample is considered to be negative and no further testing is advised. However, if the first-tier test is positive or equivocal it is recommended that second-tier testing be done. The second-tier tests are Western blot assays for both IgG and IgM antibodies. Although they also use whole cell lysates of B. burgdorferi or specific recombinant proteins as antigens, they provide higher specificity than the first-tier test due to the algorithms used for interpretation. For Lyme IgM Western blots to be considered positive, at least 2 of 3 bands (p23, p39, or p41) must be determined to be positive. And for Lyme IgG Western blots to be considered positive at least 5 of 10 bands (p18, p23, p28, p30, p39, p41, p45, p58, p66, or p93) must be read as positive. Theel, supra; CDC, supra.
Western blot assays are considered technically complex to perform and are, most often, subjective in their interpretation. Theel, supra; Molins C R, Sexton C, Young J W, Ashton L V, Pappert R, Beard C B, Schriefer M E, Collection and Characterization of Samples for Establishment of a Serum Repository for Lyme Disease Diagnostic Test Development and Evaluation, J. Clin. Microbiol. 52:3755-3762 (2014). In contrast, ELISA assays are relatively straight forward to perform, can be quantitative, and are non-subjective in their interpretation. A purpose of this study was to develop one or two recombinant antigens from B. burgdorferi that could be used in the development of a 1-tier ELISA test for the confirmation and diagnosis of LD, and that could yield overall specificities and sensitivities equal to, or better, than those of the 2-tiered method for the detection of Borrelia-specific antibodies in human sera.